Cryopreservation and vitrification are processes whereby biological specimen are preserved at subzero temperatures. Biological reactions are known to be slowered by lowering the temperature. However, said techniques must be performed carefully when living cells are involved. Indeed, water being the primarily component of living cells and their growth environment, the formation of ice crystal must be controlled during the freezing of said living cells in order to preserve their integrity.
It has been reported that freezing may cause damages to biological specimen, especially living cells. Indeed, during the freezing, ice crystals are formed, which have a serious deleterious effect on the cells. Intracellular crystals can damage the cell walls and structure, while the extracellular precipitation of water as ice crystals increases the salt concentrations to levels that can cause damages to the cells.
Therefore, compounds functioning as chemical or physical stabilizers, namely cryoprotective agents, have been widely used in order to protect cells against the stress encountered during freezing. The diffusion of the current classical cryoprotective agents such as dimethylsulfoxide (DMSO) into a cell will result in a partial replacement of intracellular water and help to prevent dehydration from ice formation during freezing.
However, DMSO is known to induce differentiation of some cells lines such as HT-60, ATCC CCL-240 (Collins et al., 1978). Furthermore, DMSO has been reported to expose cells, such as reproductive cells and embryos, to genetic transformation, which is unacceptable for cryopreservation.
More importantly, DMSO oxidation, which is highly rapid, induces the formation of toxic compounds. Finally, DMSO has been reported to be responsible for destabilization of phospholipid membranes (Anchordoguy et al, 2003).
Therefore, DMSO is currently highly unadapted for use in cryopreservation. Indeed, the toxicity of cryoprotective agents is a key limiting factor in cryobiology. The use of those agents thus may be manifested in the form of cryoinjuries.
There is thus a need for a cryopreservation technique, useful for various biological specimens, including cells such as reproductive cells and embryos and which does not lead to the damages related to toxicity of cryoprotectants, cryoinjury of the frozen biological specimen and/or genetic transformation of said specimen.